氧化壓力引起之神經退化及脊髓肌肉萎縮症之藥物開發

Drug development on oxidative stress-induced neurodegeneration and spinal muscular atrophy

許雅雲(Ya-Yun Hsu)

指導教授 ： 羅怡卿 教授

高雄醫學大學/醫學院/醫學研究所/博士(2012年)

摘要

背景：
神經退化性疾病選擇性地發生在神經細胞族群，導致神經系統漸進性地遭受破壞。而造成神經退化性疾病之機轉複雜，氧化壓力和基因變異兩大因素被認為與神經退化的發生息息相關。
目的:
本論文分為兩部份進行探討，分別為氧化壓力引起的神經毒性及基因變異造成的脊髓肌肉萎縮症之藥物開發。
實驗方法:
第一部份，在氧化壓力所引起的神經毒性模式中，我們分別利用血清移除(serum-deprivation)及過氧化氫(H2O2)來誘發神經毒性，進行藥物KMUP-1及小檗鹼(berberine; BBR)的作用及機轉之探討。第二部份，使用脊髓肌肉萎縮症病人之皮膚纖維母細胞模式探討雷公藤甲素(triptolide)對於運動神經存活蛋白表現量之影響。在動物體內試驗，則是將雷公藤甲素以腹腔注射方式投予脊髓肌肉萎縮症小鼠，觀察小鼠存活率是否能被延長及在不同組織中運動神經存活蛋白表現量之改變。
主要結果:
第一部份：利用血清移除模式使細胞產生凋亡毒性，在流式細胞儀的結果顯示，KMUP-1能抑制血清移除所導致的細胞死亡，而KMUP-1的保護作用會被一氧化氮合成酶(nitric oxide synthase; NOS)抑制劑、G型蛋白激酶(protein kinase G; PKG)抑制劑以及磷酯肌醇激酶(Phosphoinositide 3-kinase; PI3K)抑制劑所阻斷。在西方點墨法的結果則顯示，KMUP-1不只能抑制血清移除所誘導的nNOS、 sGC???﹛BPKG、p-CREB、p-Akt、Bcl-2的表現量降低，也會抑制血清移除所誘發的Bax增加。在過氧化氫處理的運動神經元細胞株NSC34中，小檗鹼(BBR)能明顯減少自由基生成及提高細胞存活率，同時也能提高抗氧化防禦系統能力及氧化敏感性蛋白表現，此結果能被磷酯肌醇激酶抑制劑所阻斷。此外，小檗鹼能藉由增加粒線體膜電位及減少耗氧率來提升粒線體功能，也能藉由增加抗凋亡蛋白和運動神經存活蛋白及減少凋亡蛋白產生，使細胞產生抗凋亡作用。第二部份：在脊髓肌肉萎縮症病人之纖維母細胞模式中，雷公藤甲素(triptolide)能有效增加運動神經存活蛋白及組成運動神經存活蛋白相關複合物之蛋白表現，也能增加核內gems數量及全長運動神經存活基因2與外顯子7缺乏運動神經存活基因2之訊息RNA比例。此外，雷公藤甲素能有效增加脊髓肌肉萎縮症小鼠中腦部、脊髓、腓腸肌之運動神經存活蛋白表現量，同時也能延長病鼠的存活率及改善體重下降之情況。
結論：
KMUP-1能有效抑制血清移除所誘發的神經細胞毒性及細胞凋死，其作用機轉包括透過活化cGMP/PKG、PI3K/Akt/CREB、Bcl-2/Bax等訊息傳遞途徑；奈米莫耳濃度下的小檗鹼能有效活化PI3K/Akt 相關之細胞保護及抗氧化途徑來達到神經保護作用；雷公藤甲素能藉由促進運動神經存活基因2之活化及增加核內gems數量來增加運動神經存活蛋白表現，同時也能延長病鼠之存活率。經由以上的研究結果，我們推論KMUP-1、小檗鹼及雷公藤甲素具有潛力應用於神經退化性疾病之改善。

關鍵字

神經退化性疾病 ； 氧化壓力 ； 脊髓肌肉萎縮症 ； KMUP-1 ； 小檗鹼 ； 雷公藤甲素

並列摘要

Background
Neurodegenerative diseases selectively target subpopulations of neurons, leading to the progressive failure of nervous system. However, the mechanisms of neurodegenerative diseases remain elusive. Oxidative stress and gene mutation are implicated in the causation of neurodegeneration. Therefore, search for efficaciously neuroprotective agents has emerged as a major drug-discovery strategy.
Purpose
This thesis focused on searching efficaciously neuroprotective agents in oxidative stress-induced neurotoxicity and spinal muscle atrophy (1).
Experimental approach
First, the effects and mechanisms of KMUP-1 and berberine (BBR) on neuroprotection were determined by serum deprivation-induced and H2O2-induced neurotoxicity, respectively. Second, the effects and mechanisms of triptolide on the production of survival motor neuron (SMN) protein were determined by cell-based assays using motor neuronal cell line NSC34 and skin fibroblasts from SMA patients. Wild-type (Smn+/+SMN2-/-, C57BL/6) and SMA-like (Smn-/- SMN2+/+) mice received triptolide (0.01 or 0.1 mg/kg/day) by intraperitoneal injection to examine the survival rate and the change in SMN protein in spinal cord and muscle tissues.
Results
In serum-deprivated SH-SY5Y cells, KMUP-1 not only enhanced expression of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclase ?? 1(sGC??1), Protein kinase G (PKG), phosphor-cAMP response element binding (p-CREB), p-Akt and B-cell lymphoma 2 (Bcl-2), but also attenuated Bcl-2-associated X protein (Bax) expression. Flow cytometric analysis using Annexin V also showed KMUP-1 increased cell viability, but lacked protective effects in the presence of nitric oxide synthase inhibitor L-NAME, PKG inhibitor Rp-8-pCPT-cGMPS or LY294002. In H2O2-treated NSC34 cells, BBR significantly attenuated reactive oxygen species (ROS) production and increased cell viability, antioxidant defense (GSH and SOD) and oxidant-sensitive proteins (HO-1 and Nrf2), which were blocked by LY294002. Furthermore, BBR improved mitochondrial function by increasing mitochondrial membrane potential and decreasing the oxygen consumption rate. BBR-induced anti‐apoptotic function was demonstrated by increasing anti-apoptotic protein (Bcl-2, SMN) and by decreasing apoptotic proteins (cytochrome c, Bax and caspase). In NSC34 cells and human SMA fibroblasts, triptolide at picromolar concentration significantly increased SMN protein expression and SMN complex component (Gemin2 and Gemin3) amounts. In human SMA fibroblasts, triptolide increased SMN-containing nuclear gems and the ratio of full length transcripts (FL-SMN2) to transcripts lacking exon 7 (SMN2Δ7). Furthermore, in SMA-like mice, triptolide significantly increased SMN protein levels of brain, spinal cord and gastrocnemius muscle. Furthermore, triptolide treatment increased survival and reduced weight loss in SMA-like mice.
Conclusions
cGMP/PKG, PI3K/Akt/CREB and Bcl-2/Bax signals play critical roles in the neuroprotective effects of KMUP-1 on serum deprivation-induced toxicity. BBR, which is active at nanomolar concentration, is a potential neuroprotective agent via PI3K/Akt-dependent cytoprotective and antioxidant pathways. Triptolide enhances SMN protein production by promoting SMN2 activation, exon 7 inclusion and increasing nuclear gems, and extends survival in SMA mice. These results suggest KMUP-1, BBR, and triptolide might be a potential strategy for neurodegenerative diseases.

並列關鍵字

neurodegenerative disease ； oxidative stress ； spinal muscular atrophy ； KMUP-1 ； berberine ； triptolide

參考文獻

1. Rommel, C.; Bodine, S. C.; Clarke, B. A.; Rossman, R.; Nunez, L.; Stitt, T. N.; Yancopoulos, G. D.; Glass, D. J. (2001) Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell Biol. 3, 1009-1013.

2. Gao, H. M.; Hong, J. S. (2008) Why neurodegenerative diseases are progressive: uncontrolled inflammation drives disease progression. Trends Immunol. 29, 357-365.

3. Coppede, F.; Mancuso, M.; Siciliano, G.; Migliore, L.; Murri, L. (2006) Genes and the environment in neurodegeneration. Biosci Rep. 26, 341-367.

4. Bonda, D. J.; Wang, X.; Perry, G.; Smith, M. A.; Zhu, X. (2010) Mitochondrial dynamics in Alzheimer's disease: opportunities for future treatment strategies. Drugs Aging. 27, 181-192.

5. Polster, B. M.; Fiskum, G. (2004) Mitochondrial mechanisms of neural cell apoptosis. J Neurochem. 90, 1281-1289.